Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding Sequence Listing and the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
A. Regulation of Apoptosis
1. Apoptosis is important in diverse physiologic processes; the abnormal regulation of apoptosis is important in diverse pathologic processes, including turmorigenesis. PA1 2. Apoptosis and the cell cycle may share common pathways. PA1 3. An evolutionarily conserved set of cellular genes regulate apoptosis. PA1 4. Bcl-2, the proto-oncogene, inhibits a variety of types of apoptosis. PA1 5. Dysregulated Bcl-2 expression occurs in a wide variety of human cancers and contributes to neoplastic cell expansion. PA1 6. The mechanism by which Bcl-2 inhibits apoptosis is still poorly understood. PA1 7. No functional links have been identified between inhibitors of apoptosis and inhibitors of cell cycle. PA1 8. Further investigation of the mechanism(s) underlaying the death repressor activity of Bcl-2, including the characterization of novel Bcl-2 interacting proteins, will provide new insights into apoptosis and diseases in which apoptosis plays a pathogenetic role. PA1 1. Several genes are responsible for inherited breast and ovarian cancer. PA1 2. Molecular genetic evidence suggests chromosome 17q21 may contain a second tumor suppressor tumor suppressor gene (in addition to BRCA1) that is important in sporadic breast and ovarian cancer.
Apoptosis is a highly conserved innate mechanism by which mammalian cells commit suicide. This mechanism allows an organism to eliminate unwanted or defective cells by an orderly process of cellular disintegration, and is characterized by certain stereotypic biochemical (e.g. endonucleosomal cleavage into 180-200 bp multimers) and morphologic features (e.g. chromatin condensation, cytoplasmic blebbing, etc.). Apoptosis plays a role in physiologic processes such as differentiation during embryogenesis, establishment of immune self-tolerance, and killing of cytotoxic immune cells, and apoptosis can be induced in response to a variety of stimuli including DNA damage, growth factor withdrawal, Ca.sup.2+ influx, ischemia, and viral infection. The unwanted occurrence of apoptosis may play a role in neurodegenerative diseases and aging, and the diminution of apoptotic death may play a role in cancer and chemoresistance.
In recent years, the concept that the cell cycle and apoptosis are inextricably linked has gained widespread support in the cell death field. Several different lines of evidence support this concept. For example, in response to different death signals, normally quiescent cells express elevated levels of cell cycle genes (Buttyan, 1991; Freeman, 1994). Oncogenes such as c-myc (Evan, et al., 1996), ras (Wyllie, et al., 1987; Tanaka, et al., 1994) and adenovirus ELA (White, 1991), that promote cell proliferation, also act as triggers of apoptosis. Loss of normal restraints at the G1 checkpoint, such as inactivation of the retinoblastoma gene product, p105Rb (Clarke, et al, 1992; Lee, et al 1992; Jacks, et al, 1992), or deregulated expression of the G1-specific E2F transcription factors (Shan, et al., 1994; Qin, et al., 1994; Wu, et al., 1994) results in uncontrolled proliferation and apoptosis. Loss of the p53 tumor suppressor gene results in resistance to certain apoptotic triggers, and p53 overexpression induces some types of apoptosis (reviewed in Evan, 1995). The morphologic features of apoptosis resemble those of mitotic catastrophe (reviewed in King and Cidlowski, 1995), and premature activation of cyclin dependent kinases is required for some forms of apoptosis (Shi, et al., 1994). Furthermore, several agents that block cell cycle progression also protect neuronal cells from apoptosis induced by withdrawal of trophic factor support (Farinelli and Greene, 1996) and T lymphocytes from apoptosis induced by T-cell receptor ligation (Boehme and Lenardo, 1993). These observations all support the notion of a link between the cell cycle and apoptosis.
Several mammalian genes have been identified that function as either inducers (e.g. faslapo-1, bax, ICE-like cysteine proteases, p53) or repressors (e.g. bcl-2, bcl-x.sub.s, bcl-x.sub.L) of an evolutionarily conserved apoptotic death pathway. Prevailing hypotheses in the cell death field are that a family of ICE-like cysteine proteases (CED-3, ICE, Nedd-2/ICH-1, CPP32) constitute the pivotal triggers of both nematode and mammalian cell suicide program and that a family of bcl-2-related genes constitute the final downstream negative regulators of cell death. Despite the identification of several effectors and repressors of cell death, the precise molecular mechanisms underlining the action of each of these genes remains poorly defined.
Bcl-2 (for B cell lymphoma 2) is the prototypic anti-apoptotic gene. It was first discovered by virtue of its involvement in the t(14:18) chromosomal translocations found in the majority of non-Hodgkin's B cell lymphomas (Tsujimoto and Croce, 1985). Bcl-2 can prevent or delay apoptosis induced by a wide variety of stimuli (reviewed in Park and Hockenbery, 1996), including growth factor deprivation, alterations in Ca.sup.2+, free radicals, cytoxic lymphokines, some types of viruses, radiation and most chemotherapeutic drugs. The ability of Bcl-2 to inhibit apoptosis induced by such diverse stimuli suggests that this oncoprotein controls a common final pathway involved in cell death regulation.
While the bcl-2 gene was first discovered because of its involvement in t(14:18) translocations found frequently in non-Hodgkin's lymphomas, high levels and aberrant patterns of bcl-2 gene expression have been reported in a wide variety of human cancers, including colorectal, gastric, prostate, non-small cell lung, neuroblastomas, breast and ovarian cancer (reviewed in Reed, et al., 1996). In these tumors, it is thought that Bcl-2 contributes to neoplastic cell expansion by preventing cell turnover caused by physiological cell death mechanisms. In addition to its role in the development of human tumors, high levels of Bcl-2 expression are thought to play an important role in the resistance of tumor cells to cytotoxic anticancer drugs and radiation.
Several potential mechanisms of action have been proposed for Bcl-2, including protection against oxidative stress (Hockenbery et al., 1993; Kane et al., 1993), regulation of intracellular Ca.sup.2+ homeostasis (Lam, et al., 1993), antagonism of cell death proteases (e.g. ICE-like family of cysteine proteases) (Miura, et al., 1993) and other cell death effectors (e.g. bax) (Yin, et al., 1994), and association with the signal transducing proteins, R-ras and Faf-1 (Fernandez-Sarbia and Bischoff, 1993; Wang, et al., 1994). In addition, two recent reports have suggested that Bcl-2 may exert anti-apoptotic effects by delaying cell cycle progression (Mazel, et al., 1996; Borner, 1996). Despite these numerous proposed mechanisms, there is considerable contradictory evidence and no universal agreement in the cell death field as to how Bcl-2 actually works. Further elucidation of the precise mechanism(s) of action of Bcl-2 is a high research priority in the field.
According to the concept that the cell cycle is linked to apoptosis, one would predict that cellular genes that inhibit apoptosis would be functionally linked to genes that exert effects on the cell cycle. Along these lines, Bcl-2 has been shown to delay cell cycle progression (as stated above), and Bcl-2 has also been postulated to function as a nuclear "gatekeeper" that regulates nuclear access of cyclin-dependent kinases. However, to date, Bcl-2 has not been shown to directly interact with any proteins that affect the cell cycle.
Understanding how Bcl-2 inhibits cell death is a critical question that has important implications for an understanding of all physiologic processes that involve cell death.
B. Molecular Pathogenesis of Breast and Ovarian Cancer
The existence of one gene predisposing to breast and ovarian cancer on chromosome 17q21, BRCA1, was proven by linkage analysis several years ago (Hall, et al., 1990), and isolated in 1994 by positional cloning (Miki, et al., 1994; Futreal, et al., 1994). BRCA1 is mutated in the germline and the normal allele is lost in tumor tissue from approximately 50% of cases of hereditary breast and ovarian cancer (reviewed in Szabo and King, 1996). BRCA2, a second breast cancer susceptibility gene, has been mapped to chromosome 13q21 and is presently implicated in 20% of hereditary cases (reviewed in Szabo and King, 1996). At least two other genes, p53 and the androgen receptor are also responsible for inherited predisposition to breast cancer in families. Other epidemiologic studies have suggested that carriers of mutations in the ataxia telangieclasia gene and HRAS1 minisatellite locus are also at increased risk of breast cancer.
Allelic deletions of chromosome 17q21 (loss of heterozygosity [LOH] that include that BRCA1 region are found to occur in 50-70% of breast carcinomas (Futreal, et al., 1992; Cropp, et al., 1993; Saito, et al., 1993) and in up to 75% of ovarian carcinomas (Russell, et al., 1991; Sato, et al., 1991; Eccles, et al., 1991; Yang-Feng, et al., 1993). However, while several studies have confirmed the role of germline BRCA1 mutations in hereditary breast and ovarian cancers (Miki, et al., 1994; Futreal, et al., 1994), somatic mutations in BRCA1 have been found in very few cases of sporadic cancers (Futreal, et al., 1994; Takahashi, et al., 1995; Merajver, et al., 1995; Hosking, et al. 1995). This raises the strong possibility that the frequent allelic loss on chromosome 17q21 in sporadic breast and ovarian cancer reflects the involvement of an additional tumor suppressor gene. In further support of this hypothesis, more detailed deletion mapping of sporadic epithelial ovarian carcinomas has revealed a common deletion unit, located on chromosome 17q21 that is located approximately 60 kb centromeric to BRCA1 (Tangir, et al., 1996). Thus, the presence of LOH in sporadic ovarian cancer cases of a region of chromosome 17q21 that does not encompass BRCA1 may reflect the presence of an additional tumor suppressor gene.
C. Overview
In response to different death signals, normally quiescent cells express elevated levels of cell cycle genes (Buttyan, R., 1991; Freeman, R. S., et al., 1994) Oncogenes, such as c-myc (Evan, G. J., 1992), ras (Wyllie, A. H., et al. (1987; Tanaka, N., et al., 1994), and adenovirus E1A (White, E., et al., 1991), that promote cell proliferation, also act as triggers of apoptosis. Loss of these restraints at the G1 checkpoint, such as inactivation of the retinoblastoma gene product, p105Rb (Clarke, A. R., et al., 1992; Lee, E-H., et al. (1992; Jacks, T., et al., 1992), or deregulated expression of the G1-specific E2F transcription factors (Shan, B., et al., 1994; Qin, X., et al. 1994; Wu, X. and Levine, A. J., 1994), results in uncontrolled proliferation and apoptosis. The p53 tumor suppressor gene enforces cell cycle arrest or apoptosis following DNA damage and also mediates apoptosis triggered by a variety of non-genotoxic stimuli (reviewed in Evan, G. I., et al., 1995). The morphologic features of apoptosis resemble those of the mitotic catastrophe (reviewed in King, K. L., et al., 1995), and premature activiation of cyclin-dependent kinases is required for some forms of apoptosis (Shi, L., et al., 1994). Furthermore, phamacologic and genetic inhibitors of cell cycle progression protect many cell types from apoptosis (Farinelli, S. E., et al., 1996; Meikrantz, W. and Schlegel, R., 1996). These observations all suppport the notion that regulation of the cell cycle is intricately involved in cell death.
According to this notion, cellular genes that inhibit apoptosis may be functionally linked to genes that exert effects on the cell cycle. Consistent with this hypothesis, the death-protective activity of Bcl-2, the prototype member of a family of cell death regulators, is associated with a delay in the kinetics of cells cycle progression (Borner, C., 1996; Mazel, S., et al., 1996; O'Reilly, L. A., et al., 1996; Vairo, B., et al. 1996). Furthermore, Bcl-2 overexpression suppresses NF-AT signalling and activation of T-cells (Linette, G. P., et al., 1996; Shibasaki, F., et al., 1997). Such findings suggest that regulators of cell death can affect cellular proliferation pathways. However, it is, as yet unknown, whether interactions between cell death regulators and cellular proliferation pathways are mechanistically important in the death repressor activity of anti-apoptotic genes.
To further understand the mechanism by which bcl-2 protects against apoptosis, the yeast hybrid system was used to screen a mouse brain library for complementary cDNAs encoding proteins that bind to Bcl-2. The yeast two hybrid system has proven useful to identify interactions between Bcl-2 and novel proteins (Fernandez-Sarbia, M. J., et al., 1993; Boyd, J., et al., 1994; Yang, E., et al., 1995; Farrow, S. N., et al., 1995), to analyze hetero-and homodimerization of Bcl-2 family members (Sato, T., et al., 1994; Sedlack, T. W., et al., 1995), and to perform structure-function analysis of the Bcl-2 protein.